GluN2C selective inhibition is a target to develop new antiepileptic compounds.

OBJECTIVE: Many early-onset epilepsies present as developmental and epileptic encephalopathy associated with refractory seizures, altered psychomotor development, and disorganized interictal cortical activity. Abnormal upregulation of specific N-methyl-d-aspartate receptor (NMDA-R) subunits is being disentangled as one of the mechanisms of severe early-onset epilepsies. In tuberous sclerosis complex (TSC), upregulation of the GluN2C subunit of the NMDA-R with slow deactivation kinetic results in increased neuronal excitation and synchronization. METHODS: Starting from an available GluN2C/D antagonist, NMDA-R-modulating compounds were developed and screened using a patch clamp on neuronal culture to select those with the strongest inhibitory effect on glutamatergic NMDA currents. For these selected compounds, blood pharmacokinetics and passage through the blood-brain barrier were studied. We tested the effect of the most promising compounds on epileptic activity in Tsc1+/- mice brain slices with multielectrode array, and then in vivo at postnatal ages P14-P17, comparable with the usual age at epilepsy onset in human TSC. RESULTS: Using a double-electrode voltage clamp on isolated NMDA currents, we identified the most prominent antagonists of the GluN2C subunit with no effect on GluN2A as a means of preventing side effects. The best compound passing through the blood-brain barrier was selected. Applied in vivo in six Tsc1+/- mice at P14-P17, this compound reduced or completely stopped spontaneous seizures in four of them, and decreased the background activity disorganization. Furthermore, ictal-like discharges stopped on a human brain sample from an infant with epilepsy due to TSC. INTERPRETATION: Subunit-selective inhibition is a valuable target for developing drugs for severe epilepsies resulting from an upregulation of NMDA-R subunit-mediated transmission.

Epilepsy in young Tsc1(+/-) mice exhibits age-dependent expression that mimics that of human tuberous sclerosis complex.

OBJECTIVE: To describe the epileptic phenotype of Tsc1(+/-) mice pups in comparison with age-related seizures in human tuberous sclerosis complex (TSC). METHODS: Tsc1(+/-) and control mice underwent intracranial electroencephalography (EEG) recording at postnatal ages (P)8 to P33, with linear silicon probe implanted in the somatosensory cortex of one or both hemispheres for 8-24 h. Ictal events were classified visually by independent analyzers; distinct EEG patterns were related to age and analyzed to quantify field potential characteristics and signal dynamics between hemispheres. We collected retrospectively 20 infants with prenatally diagnosed TSC and EEG before seizure onset, and analyzed the electroclinical course of epilepsy, taking into account a first-line treatment by vigabatrin. RESULTS: Spontaneous seizures were disclosed in 55% of Tsc1(+/-) mice at P9-18. Three ictal patterns were identified: from P9 to P12 "spike clusters" consisted of recurring large spikes without clinical correlate; "spasm-like" discharges dominated from P13 to P16 consisting of high amplitude large field potential superimposed with or followed by fast activity repeated every 2-10 s for at least 20 s, accompanied by rhythmic limb contractions; from P14 to P18 a "tonic-clonic like" pattern comprised rhythmic spikes of increasing amplitude with tonic-clonic movements. Early onset "spike clusters" were mainly unilateral, whereas "spasm-like" and "tonic-clonic like" patterns were bilateral. Interhemispheric propagation was significantly faster for "tonic-clonic like" than for "spasm-like" events. In infants diagnosed prenatally with TSC, clusters of sharp waves or spikes preceded the first seizure, and vigabatrin prevented the development of seizures. Patients treated after seizure onset developed spasms or focal seizures that were pharmacoresistant in 66.7% of cases. SIGNIFICANCE: Tsc1(+/-) mice pups exhibit an age-dependent seizure pattern sequence mimicking early human TSC epilepsy features. Spike clusters before seizure onset in TSC should be considered as a first stage of epilepsy reinforcing the concept of preventive antiepileptic therapy.

Seizures and epilepsy in hypoglycaemia caused by inborn errors of metabolism.

AIM: The aim of the study was to characterize seizures and epilepsy related to hypoglycaemia. METHOD: We analyzed the files of 170 consecutive patients referred for hypoglycaemia (onset 1h to 4y) caused by inborn errors of metabolism (glycogen storage disease type I, fatty acid ß-oxidation disorders, and hyperinsulinism). RESULTS: Ninety patients (42 males and 48 females; 38 neonates and 52 infants/children) had brief hypoglycaemic seizures (68%) or status epilepticus (32%). Status epilepticus occurred earlier (mean 1.4d) than brief neonatal seizures (4.3d, p=0.02). Recurrent status epilepticus followed initial status epilepticus and was often triggered by fever. Epilepsy developed in 21 patients. In 18 patients, epilepsy followed hypoglycaemic status epilepticus and began with shorter delay when associated with grey matter lesions (1.9mo, standard error of the mean [SEM] 1mo) than with white matter damage (3.3y [SEM 1y], p=0.003). Three patients with hyperinsulinism developed idiopathic epilepsy following brief neonatal seizures. INTERPRETATION: Brief neonatal hyperinsulinaemic hypoglycaemic seizures have characteristics of idiopathic neonatal seizures. Neonatal status epilepticus should be prevented by the systematic measurement of glucose blood level. Recurrent seizures never consist of status epilepticus when following brief initial seizures. Epilepsy is symptomatic of brain damage with shorter delay in the case of grey rather than white matter lesions, except in a few idiopathic cases in which epilepsy and hyperinsulinism may share a common genetic background.

Death following consumption of MDAI and 5-EAPB.

5-(2-ethylaminopropyl)benzofuran (5-EAPB) and 5,6-methylenedioxy-2-aminoindane (MDAI) are two new psychoactive substances (NPS) exhibiting MDMA-like properties. In this paper, we report the case of a 28-years old man, known as drug addict, found dead at home, with two unidentified powders next to him. External examination by the forensic pathologist was unremarkable but no autopsy was performed. Powders, blood and urine (which were the only samples available) were submitted to general unknown screening by high pressure liquid chromatography with a diode array detector (HPLC-DAD) and ultra high pressure liquid chromatography with a time-of-flight detector (UPLC-TOF-MS), after liquid-liquid extraction for biological samples, or simple dilution for powders. Analysis revealed 68% of MDAI in one powder and 87% of 5-EAPB in the other one. Significant levels of the same substances were found in blood (MDAI: 2.09 mg/L and 5-EAPB: 6.45 mg/L). The cause of death was therefore attributed to the consumption of these NPS since screening for other drugs of abuse and for alcohol was negative (oxazepam was found in urine only). 5-methylaminopropylbenzofuran (5-MAPB) and 5-aminopropylbenzofuran (5-APB) were also found in blood (0.089 and 0.546 mg/L, respectively) and urine (1.00 and 4.88 mg/L, respectively). In addition to the inherent complexity of NPS identification by itself, another analytical difficulty in this case was the identification of the EAPB positional isomer. Our routine screening methods were not able to distinguish the positional isomer, but an additional classical gas chromatography technique was able to make the distinction. Anyway, in our case, this issue was simplified thanks to the availability of a relatively pure powder that was analyzed by nuclear magnetic resonance (NMR).

Site-, Technique-, and Time-Related Aspects of the Postmortem Redistribution of Diazepam, Methadone, Morphine, and their Metabolites: Interest of Popliteal Vein Blood Sampling.

Sampling site, technique, and time influence postmortem drug concentrations. In 57 cases, we studied drug concentration differences as follows: subclavian vein-dissection/clamping versus blind stick, femoral vein-dissection/clamping versus blind stick, right cardiac chamber, and popliteal vein-dissection and clamping only. Cases were distributed in group #1 (all cases with both techniques), group #2 (dissection/clamping), and group #3 (blind stick). Sampled drugs were diazepam, methadone, morphine, and their metabolites. To assess PMR, mean concentrations and ratios were calculated for each group. Time-dependent variations of blood concentrations and ratios were also assessed. Results indicate that site, method, and time may influence postmortem distribution interpretation in different ways. Popliteal blood seems less subject to PMR. In conclusion, our study is the first to evaluate concurrently three main aspects of PMR and confirms that the popliteal vein may represent a site that is more resistant to the changes seen as a result of PMR.

Postmortem Concentration and Redistribution of Diazepam, Methadone, and Morphine with Subclavian and Femoral Vein Dissection/Clamping.

Postmortem redistribution (PMR) concerns blood drug concentration variations after death, depending on many factors such as sampling site and technique. In our study, we focused on sampling method. 30 cases were sampled, each at cardiac, subclavian, femoral, and popliteal sites. Targeted substances were diazepam, methadone, and morphine. Blind stick and dissection/clamping techniques were concomitantly performed at subclavian and femoral sites. Subclavian and femoral concentrations were compared according to technique used. To assess the influence of sampling technique on PMR, central/peripheral ratios were calculated depending on sampling method. Results show that drug concentrations tend to be lower when drawn from a clamped subclavian or femoral vein whereas ratios including subclavian and/or femoral blood concentration are influenced according to the technique used. In conclusion, clamping a subclavian or femoral vessel before sampling tends to result in lower drug concentrations and may influence ratios, suggesting the importance of isolating vessels from thoraco-abdominal viscera.

Popliteal Vein Blood Sampling and the Postmortem Redistribution of Diazepam, Methadone, and Morphine.

Postmortem redistribution (PMR) refers to the site- and time-related blood drug concentration variations after death. We compared central blood (cardiac and subclavian) with peripheral blood (femoral and popliteal) concentrations of diazepam, methadone, and morphine. To our knowledge, popliteal blood has never been compared with other sites. Intracardiac blood (ICB), subclavian blood (SB), femoral blood (FB), and popliteal blood (PB) were sampled in 30 cases. To assess PMR, mean concentrations and ratios were compared. Influence of postmortem interval on mean ratios was also assessed. Results show that popliteal mean concentrations were lower than those for other sites for all three drugs, even lower than femoral blood; mean ratios suggested that the popliteal site was less subject to PMR, and estimated postmortem interval did not influence ratios except for diazepam and methadone FB/PB. In conclusion, our study is the first to explore the popliteal site and suggests that popliteal blood is less prone to postmortem redistribution.